Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates

Matrix metalloproteinase (MMP) family members are involved in the physiolog ical remodeling of tissues and embryonic development as well as pathologica l destruction of extracellular matrix components. To study the mechanisms o f MMP action on collagenous substrates, we have constructed homotrimeric, f luorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site i n type II collagen. The substrates were designed to incorporate the fluorop hore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitr ophenyl (Dnp) in the P-5 and P-5' positions, respectively. In addition, Arg was incorporated in the P-2' and P-8' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(M ca)-Gly-Pro-Gln-Gly similar to Leu-Arg-Gly-Gln-Lys(Dnp)- Gly-Ile/Val-Arg. T wo fluorogenic substrates were prepared, one using a covalent branching pro tocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THP s were hydrolyzed by MMP-1 at the Gly similar to Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K-M = 61.2 muM. S ubsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta (243-450))], and a C-termina l domain-deleted MMP-3 [MMP-3(Delta (248-460))]. The order of k(cat)/K-M va lues was MMP-13 > MMP-1 similar to MMP-1(Delta (243-450)) similar to MMP-2 >> MMP-3 similar to MMP-3(Delta (248-460)). Studies on the effect of temper ature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation e nergies between these two substrates differed by 3.4-fold, similar to the d ifference in activation energies for MMP-1 hydrolysis of type I collagen an d gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the inv estigation of collagenase triple-helical activity.